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Cytogenetics

Processing

The processing phase of Fluorescence In Situ Hybridization (FISH) transforms a prepared slide into an analyzable image. It involves a sequence of thermodynamic and chemical manipulations designed to melt the DNA strands, allow specific probe binding, and wash away non-specific background noise. While the core workflow (Denature \(\rightarrow\) Hybridize \(\rightarrow\) Wash) is universal, specific specimen types like plasma cells or solid tissues (FFPE) require specialized pre-processing to ensure success

Core FISH Workflow

  • Denaturation (The Melt)
    • Goal: Convert double-stranded DNA (dsDNA) of both the specimen and the probe into single strands (ssDNA) so they can pair up
    • Mechanism: Heat breaks hydrogen bonds, while Formamide is used chemically to lower the melting temperature (\(T_m\)) to a range that protects cellular morphology (\(\sim 72^\circ\text{C}\)–\(75^\circ\text{C}\))
    • QC: Under-denaturation results in “refractile” nuclei and no signal. Over-denaturation results in “ghostly” nuclei and DNA destruction
  • Hybridization (The Annealing)
    • Goal: Allow the fluorescent probe to bind specifically to its complementary target sequence
    • Mechanism: Occurs at \(37^\circ\text{C}\) (typically overnight) in a humidity chamber to prevent evaporation
    • Specificity: Cot-1 DNA is added to block repetitive sequences (Alu repeats), preventing the probe from binding randomly across the genome
  • Postwash (The Cleanup)
    • Goal: Remove excess probe that is loosely bound to non-target DNA or the slide surface
    • Stringency: The strictness of the wash is controlled by Temperature and Salt Concentration
      • High Stringency: High Temp / Low Salt. Removes weak, non-specific binding
      • Low Stringency: Low Temp / High Salt. Allows mismatched binding (high background)
    • Standard: Usually 0.4x SSC at \(72^\circ\text{C}\)
  • Counterstain (The Map)
    • Goal: Visualize the nuclei/chromosomes to provide context for the probe signals
    • Reagent: DAPI (Blue) binds to AT-rich DNA
    • Mounting Medium: Contains Antifade agents (scavengers) to prevent the fluorescent dye from photobleaching (fading) under the microscope light

Specialized Processing: Plasma Cell Enrichment

Used specifically for Multiple Myeloma bone marrow specimens due to low proliferation and low tumor burden

  • Target: CD138 (Syndecan-1), a surface antigen expressed strongly on plasma cells but not on other marrow cells
  • Method (MACS): Magnetic Activated Cell Sorting
    • Marrow is mixed with anti-CD138 magnetic microbeads
    • Suspension is passed through a magnetic column
    • Plasma cells stick; normal cells flow through
    • Plasma cells are eluted to create a pure populations
  • Benefit: Increases the detection rate of abnormalities [e.g., t(4;14), del(17p)] from ~30% to >90%

Specialized Processing: FFPE Tissue Sections

Used for solid tumors (Breast, Lung, Colon) where tissue is fixed in formalin and embedded in paraffin wax. Requires aggressive Pretreatment

  • Deparaffinization: Removal of hydrophobic wax using Xylene and alcohol rehydration so aqueous probes can penetrate
  • Pretreatment (Heat): Boiling slides in buffer to break Formalin-induced protein crosslinks
  • Protease Digestion: Incubation with Pepsin to digest the protein scaffold and expose the nucleus
    • Critical Balance: Under-digestion = no probe entry. Over-digestion = tissue destruction/loss of morphology
  • Artifacts: Unlike whole cells, tissue sections suffer from Truncation (sliced nuclei causing false monosomy) and Nuclear Overlap (stacked nuclei causing false polysomy)